We thank Mark Vermeij, Kristen Marhaver, and the CARMABI Research Foundation in Cura&#231;ao for providing resources, support, and insight for this project. We thank the NAVFAC Atlantic Vieques Restoration Program and the Jacobs Engineering team for their substantial logistical and technical support in installing, maintaining, and monitoring the Coral Arks in Vieques. We are also grateful to Mike Anghera, Toni Luque, Cynthia Silveira, Natascha Varona, Andres Sanchez-Quinto, Lars ter Horst, and Ben Darby for their help and constructive input in the field. This research was funded by a Gordon and Betty Moore Foundation Aquatic Symbiosis Investigator Award to FLR &#60;https://doi.org/10.37807/GBMF9207&#62; and by the Department of Defense Environmental Security Technology Certification Program (RC20-5175).

The authors have no competing financial interests or other conflicts of interest.

The representative results presented above demonstrate that Coral Arks provide a habitat and improved water quality conditions for assembling reef communities on stable, in situ research platforms. Arks and seafloor control sites at the same depth displayed consistently different water quality profiles. Higher average current speeds and further distance from the coast reduced sedimentation and turbidity in the midwater environment at the Arks sites (Figure 6B), likely contributing to the lower measured dissolved organic carbon concentrations on the Arks (Figure 6F). Further, these improvements in water clarity resulted in elevated daytime light intensities on the Arks relative to the control sites (Figure 6A). Lower diel fluctuations in dissolved oxygen indicate improved oxygen availability for corals on the Arks compared to the benthos, especially at night (Figure 6G). These metrics have all been associated with improvements in coral survival42, growth43,44,45, and recovery from stress46,47 in past work and may be linked to enhanced survival outcomes of corals translocated to Arks as compared to benthic control sites (Figure 8). The fact that these conditions persist even after the accumulation of substantial biomass through biofouling indicates that natural recruitment processes do not diminish the improved water quality characteristics of the midwater environment. Arks were deployed 3 km offshore of the benthic control sites and likely benefitted from decreased inputs of terrestrially derived sediment, nutrients, and possibly fishing pressures that challenge nearshore sites. Siting Arks in areas with clean water and low human impact (such as offshore) may provide a better setting than heavily impacted coastal zones to propagate reef biodiversity for mesocosm-level experiments.
The preliminary findings also suggested that the midwater Arks experienced less microbialization, a central reef process associated with the degradation of benthic reef habitats4,48. High nutrient inputs and overfishing have been identified as drivers of reef-wide trophic feedback loops in which energetically destabilized microbial communities proliferate, resulting in the respiratory drawdown of metabolically available oxygen and the increased incidence of coral pathogens at the benthos6,49,50,51. The reduced abundance of free viruses on microbialized reefs, which serve as a primary lytic control on microbial community growth, indicate a breakdown in the trophic structure that favors further microbial expansion52. Water column-associated microbes on the Arks were both less abundant (Figure 7B) and physically smaller (Figure 7D) than at the seafloor sites. The Arks also displayed higher virus-to-microbe ratios (Figure 7A), abundance of free viruses (Figure 7C), and dissolved oxygen availability, particularly at night (Figure 6G). Taken together, these findings indicate that the midwater environment displayed less potential for microbialization relative to the seafloor sites. Arks, as mesocosms on which environmental conditions can be altered simply by vertical adjustment in the water column, offer an opportunity to mitigate and further explore the microbial and molecular mechanisms of reef degradation.
Geodesic spheres of two different frequencies were selected for the design of the Coral Arks presented here (Figure 1). Geodesic frequency (1V, 2V, 3V) indicates the number of repeating sub-elements in a geodesic sphere, with higher frequencies corresponding with a higher number of triangular sub-elements. From a structural perspective, geodesic polyhedra distribute mechanical stress throughout the structure, resulting in a high innate strength for their size53,54. These characteristics provide high durability and longevity but come at the cost of higher hydrodynamic drag, which can result in higher loadings on the mooring system. From a habitat perspective, the drag generated by an Ark system represents an indicator of the diffusion of momentum within the structure and, thus, the degree to which the internal ambient flow is reduced. The modeled and experimentally validated results indicate a 40%-70% reduction in the flow speed inside of the &#34;Shell&#34; Arks relative to the surrounding flow field due to the generation of turbulent flow inside the structures (see Section 6 of Supplemental File 1). While the optimal level of internal flow reduction is not clear (and differs with geodesic frequency), areas of reduced flow within the structure are important for creating niche habitats55,56, remineralizing nutrients57,58, and promoting the retention and settlement of larvae59,60. In general, larger and higher frequency geodesic structures, particularly at more exposed installation sites, require anchoring systems with higher holding power and more redundancy incorporated into the structural design.
The results from the field-based measurements of the drag component of tension on the &#34;Shell&#34; Ark mooring system closely matched those results generated from the modeled and experimental towing estimates (Figure 4) and were well within the expected design ranges. These results indicate that the assumptions of the hydrodynamic model are valid and that the model can predict drag forces over the background current ranges. However, while the deviations in the modeled and experimental data were small, the range of flows during the testing period, which were typical of ambient, non-storm flow speeds at the site, did not enable a rigorous validation over the full modeling spectrum. In predicting the design requirements of Coral Arks systems, modeling efforts should be combined with information on storm frequency and exposure at the planned deployment sites to design structures and mooring systems that can survive the anticipated hydrodynamic forces. The modeling work presented here can be used to design Ark systems at other sites with minimal inputs (desired Ark size, frequency, and average current speeds at the deployment site) by providing drag coefficients and maximum expected forces on the mooring and anchoring system.
Arks and ARMS systems are modular and may be built at different scales and with alternative materials than those described here. Although their ultimate longevity has not yet been determined, Coral Arks were designed to have an approximately 10 year life cycle. The material composition of the Arks and ARMS affects the longevity of the structures, the weight of the systems, and, therefore, the required buoyancy to offset the weight and may affect the response of early fouling communities (Supplemental File 1-Figure S7). For example, limestone provides a more natural substrate for biological colonization on the ARMS and is readily and inexpensively sourced on most carbonate reef islands, but it is more fragile and heavier than other materials such as PVC and fiberglass. These factors should be considered against site-specific characteristics to design ARMS, Arks, and mooring systems that best address the desired project outcomes.
The deployment sites for Coral Arks should also be selected based on the intended project goals (i.e., research, mitigation, or restoration). Factors to consider for site selection include the access to materials, reef state or condition, community investment/involvement, resource limitation, institutional support, and permit requirements. Coral Arks may provide opportunities to meet specific needs at sites that (1) contain living coral reefs that are in relatively poor condition and would benefit from restoration activities to enhance the coral recruitment, coral cover, coastal protection, or human food resources; (2) have a need for the translocation of corals to another location, which may occur, for example, when there are legal requirements to move living corals off of debris items slated for removal (at these sites, Coral Arks can be used in collaboration with, or in support of, existing restoration and outplanting efforts to improve translocation outcomes); (3) require research into novel conservation and restoration technologies using Coral Arks to improve the success of local efforts; or (4) have sufficiently distinct local conditions (i.e., different magnitude of anthropogenic impact), meaning standardized mesocosms could yield meaningful comparisons about reef processes and interventions. The specific approaches for monitoring aspects of the Coral Arks ecosystem such as biological growth, diversity, and water chemistry will vary between projects based on the project goals and site-specific variables.A representative outline for the scientific monitoring of Coral Arks conducted to date is provided in Section 5 of Supplemental File 1.
The design of Coral Arks structures can accommodate corals of nearly any species, size, and age and should provide improved conditions relative to those on a disturbed reef benthos. Depending on the growth and calcification rates observed on a given system, the addition of positive buoyancy to the Arks structures may be required to compensate for biological growth and to reduce the risk of sinking. Positively buoyant midwater structures can be weighed using a tension/compression load cell, or strain gauge, to determine if the in-water weight of the community is increasing (Figure 5). Periodic or long-term measurements using the load cell can complement other finer-resolution coral growth metrics to generate a metric of community-level growth/calcification and have been included as a regular maintenance task to determine if the system has sufficient positive buoyancy to compensate for this biological growth over time. In the case that an installed Ark can no longer be monitored or maintained, it could be relocated and/or the buoyancy could be removed to allow the Ark to be firmly attached to the benthos.
The methods described here provide researchers with a versatile toolkit for assembling midwater reef communities that can be sited at locations with improved water quality. By altering the depth or location of the Arks structures, changes in water quality parameters can be experimentally linked to changes in reef community structure and successional trajectories. This design feature allows researchers to exploit the abundant and underutilized space in the midwater environment to assemble and study coral reef mesocosms. The use of seeded ARMS to translocate cryptic biodiversity and deliver a &#34;boost&#34; to the natural recruitment of mobile grazing invertebrates provides a functional solution for reducing algal biofouling and, thus, benthic competition for corals. Using established and standardized sampling structures as components of this system provides added value by enabling the long-term monitoring of cryptic communities on Arks and comparison to datasets generated using ARMS as a global biodiversity census tool.
Coral Arks can serve as a more holistic, integrated, and self-regulating platform for propagating coral and invertebrate biomass that can then be outplanted to nearby degraded reefs and can provide a safe haven for corals to grow and reproduce in improved water quality conditions. As is currently being demonstrated in Puerto Rico, Arks can yield improved survival outcomes for mitigation projects involving the relocation of corals and reef biodiversity from debris items or degraded areas. Arks have relevance in long-term projects as a method to replace habitats for fish populations, test novel conservation strategies, and preserve native reef biodiversity. In the process, Arks provide versatile tools for conducting in situ studies of reef assemblies and ecological succession and may generate novel insights into reef connectivity.

Across the globe, coral reef ecosystems are undergoing transitions from high-biodiversity, coral-dominated benthic communities to lower-diversity communities dominated by turf- and fleshy macroalgae1,2,3. Decades of progress in characterizing the mechanisms of coral reef degradation have revealed how links between microbial and macro-organismal communities enhance the pace and severity of these transitions. For example, the overfishing of reefs by human populations initiates a trophic cascade in which excess photosynthetically derived sugars from ungrazed algae shunt energy into the reef microbial communities, thus driving pathogenesis and causing coral decline4,5,6. This trophic downgrading is reinforced by the loss of biodiversity on reefs that results from water quality decline7,8. Mesocosm-level experiments can be used to better understand and mitigate the trophic downgrading of coral reef communities by enhancing biodiversity and improving water quality, but logistical challenges make these studies difficult to implement in situ.
A consequence of trophic downgrading on reefs is the widespread loss of cryptic biodiversity, much of which remains uncharacterized7,9. Corals rely on a diverse suite of cryptic reef organisms (&#34;cryptobiota&#34;) that support their health by playing integral roles in predator defense10, cleaning11, grazing competing algae12,13, and the regulation of reef water chemistry14,15. Until recently and due to the methodological limitations of visual surveys, reef cryptobiota have been underrepresented and poorly understood in the context of reef ecology, and they are, thus, rarely considered in efforts to restore or rebuild reefs. In the past decade, the use of standardized settlement units called Autonomous Reef Monitoring Structures (ARMS) combined with high-throughput sequencing approaches has enabled the better collection and characterization of reef cryptobiota16,17. ARMS passively recruit representatives of almost all known coral reef biodiversity and have helped reveal numerous functional roles of cryptic organisms in reef-scale processes9,18,19,20,21,22,23. These settlement units, therefore, provide a mechanism to translocate cryptic reef biota alongside corals in order to assemble more intact reef communities with biologically mediated mechanisms, such as grazing, defense, and enhancement of local water quality, that are essential to maintaining the trophic structure.
Coral-dominated reefs thrive in high-light, low-nutrient, and well-oxygenated environments. Human activities such as urbanization, agriculture, and overfishing have reduced the water quality on many coral reefs by increasing the sediment, nutrients, metals, and other compounds in runoff24,25 and by altering biogeochemical cycling26. In turn, these activities degrade reef communities through smothering, energy depletion, the delivery of pollutants associated with sedimentation27,28, enhancing the growth of macroalgae that compete with corals29, increasing the abundance of microbial pathogens6,30,31, and creating hypoxic zones that kill cryptic invertebrates32,33. These and other &#34;local impacts&#34; are compounded by regional and global changes in ocean conditions, including increasing temperatures and decreasing pH, further worsening the conditions for corals and other reef organisms34,35. At the benthic-water interface, specifically, the respiratory and photosynthetic dynamics of benthic communities cause diel fluctuations in the pH and dissolved oxygen, which become more pronounced on highly degraded reefs, thus creating conditions that benthic invertebrates cannot tolerate32,36,37,38. Providing appropriate water quality conditions is, therefore, essential for assembling functioning reef communities, but this remains challenging because an increasing number of reefs are trapped in various states of degradation.
Many of the challenges faced by corals and foundational cryptic taxa on the benthos may be overcome via relocation to the midwater, defined here as the water column setting between the ocean surface and the seafloor. In the midwater environment, water quality is improved39,40, sedimentation is reduced, and the distance from the seafloor dampens fluctuations in the parameters associated with benthic metabolism. These characteristics are improved further by moving offshore, where land-based anthropogenic impacts, such as terrestrially derived runoff, become increasingly diluted with distance from the coast. Here, we introduce and provide protocols to build, deploy, and monitor Coral Reef Arks, an approach that leverages improved water quality conditions in the midwater and incorporates cryptic biodiversity on anchored, positively buoyant structures for the assembly of coral reef communities.
Coral Reef Arks systems, or &#34;Arks,&#34; are comprised of two primary components: (1) a suspended rigid geodesic platform elevated above the benthos and (2) organism-covered or &#34;seeded&#34; ARMS that translocate reef cryptobiota from nearby benthic areas, thereby supplementing the natural recruitment processes to provide the translocated corals with a more diverse and functional reef community. A geodesic structure was selected to maximize the strength and minimize the building material (and, thus, the weight), as well as to create an internal, turbulent flow environment analogous to the reef matrix.
Two designs of Arks were successfully installed at two Caribbean field sites and are currently being used for research into reef community establishment and ecological succession (Figure 1). Coral Arks structures are intended to be long-term research platforms, and as such, a primary focus of this manuscript is to describe protocols to site, install, monitor, and maintain these structures to maximize their stability and longevity in the midwater environment. A combination of modeling and in-water testing was used to evaluate the drag characteristics of the structures and adjust the design to withstand the anticipated hydrodynamic forces. After installation, reef communities were established on the Arks and on nearby benthic control sites at the same depth through a combination of active translocation (corals and seeded ARMS units) and natural recruitment. Water quality conditions, microbial community dynamics, and coral survival on the Arks were documented at several time points throughout the early successional period and compared against the benthic control sites. To date, the conditions associated with the midwater Coral Arks environment have been consistently more favorable for corals and their associated cryptic consortia relative to the neighboring benthic control sites at the same depths. The methods below describe the steps required to replicate the Coral Arks approach, including how to select sites and design and deploy Coral Arks structures. Suggested approaches for monitoring Coral Arks are included in Supplemental File 1.

coral reef arks: an in situ mesocosm and toolkit for assembling reef communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions that include high light levels, rapid water flow, and low nutrient levels. Poor water quality and other anthropogenic stressors have caused coral mortality in recent decades, leading to trophic downgrading and the loss of biological complexity on many reefs. Solutions to reverse the causes of trophic downgrading remain elusive, in part because efforts to restore reefs are often attempted in the same diminished conditions that caused coral mortality in the first place. 
Coral Arks, positively buoyant, midwater structures, are designed to provide improved water quality conditions and supportive cryptic biodiversity for translocated and naturally recruited corals to assemble healthy reef mesocosms for use as long-term research platforms. Autonomous Reef Monitoring Structures (ARMS), passive settlement devices, are used to translocate the cryptic reef biodiversity to the Coral Arks, thereby providing a "boost" to natural recruitment and contributing ecological support to the coral health. We modeled and experimentally tested two designs of Arks to evaluate the drag characteristics of the structures and assess their long-term stability in the midwater based on their response to hydrodynamic forces. 
We then installed two designs of Arks structures at two Caribbean reef sites and measured several water quality metrics associated with the Arks environment over time. At deployment and 6 months after, the Coral Arks displayed enhanced metrics of reef function, including higher flow, light, and dissolved oxygen, higher survival of translocated corals, and reduced sedimentation and microbialization relative to nearby seafloor sites at the same depth. This method provides researchers with an adaptable, long-term platform for building reef communities where local water quality conditions can be adjusted by altering deployment parameters such as the depth and site.

Watch this Scientific Journal Video about Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities at JoVE.com

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore.

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Coral reefs thrive and provide maximal ecosystem services when they support a multi-level trophic structure and grow in favorable water quality conditions ...

coral-reef-arks-an-situ-mesocosm-toolkit-for-assembling-reef

Research

JoVE Journal

Environment

3.2K Views.  San Diego State University. Koraalarken kunnen belangrijke vragen beantwoorden over hoe mariene gemeenschappen zich verzamelen, in de loop van de tijd veranderen en reageren op veranderende omgevingsomstandigheden, inclusief hoe de biologie de abiotische omgeving verandert. Koraalarken bieden een repliceerbaar, schaalbaar en verticaal instelbaar onderzoeksplatform voor het bouwen en experimenteren met koraalrifgemeenschappen in hun natuurlijke omgeving en op ecosysteemschaal. Deze methode om ARMS en arken te combineren ...

Video: Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Bioengineering

A Combined Micro-Device System for Promoting and Monitoring Coral Growth

An Integrated Micro-Device System for Coral Growth and Monitoring

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of the coral culture environment is highly in demand for coral conservation and study. Here, we developed a semi-closed coral culture micro-device system as a multi-functional platform, which can provide accurate and programmable temperature control, a sterile initial environment, long-term stable water quality, an adjustable dissolved oxygen concentration, and a customized light spectrum for corals. Owing to the modular design, the coral culture system can be upgraded or modified by installing desirable new modules or removing existing ones. Currently, under appropriate conditions and with proper system maintenance, the sample corals can survive for at least 30 days in a healthy state. Furthermore, due to the controllable and sterile initial environment, this coral culture system can support research into the symbiotic relationship between corals and associated microorganisms. Therefore, this micro-device system can be applied to monitor and investigate sea corals in a relatively quantitative manner.

Author Spotlight: Advancing Coral Culture - Creating a Semi-Quantitatively Controlled Microenvironment System to Counter Current Limitations 

This protocol describes the development of a modular controllable micro-device system that can be applied for the long-term culturing and monitoring of sea corals.

Corals are fundamental organisms in marine and coastal ecosystems. With the advancement of coral protection research in recent years, precise control of ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Biology

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Physiological Characterization of the Coral Holobiont Using a New Micro-Respirometry Tool

Metabolic activity, defined as the sum of organismal processes that involve energy, is of critical importance in understanding the function and evolution of life on earth. Measuring organismal metabolic rates is, therefore, at the center of explaining the physiological states of organisms, their ecological roles, and the impact of environmental change on species within terrestrial and aquatic ecosystems. On coral reefs, measures of metabolism have been used to quantify symbiosis functioning between corals and their obligate algal symbionts (Symbiodiniaceae), as well as assess how environmental stressors, including climate change, will impact coral health. Despite this significance, there is a lack of methods, and therefore data, relating to metabolic rate measurements in coral offspring, likely due to their small size. To address this gap, this study aimed to develop a custom setup for measuring the respiration of small (millimeter size range) marine animal ecologies. This low cost and easy setup should allow for the improved measurement of metabolic rate. This will be essential for applied ecological research utilizing the sexual production of corals for reef restoration.

This protocol describes the setup and running of a micro-respirometry system that can be employed to investigate the physiological features of the coral holobiont.

Metabolic activity, defined as the sum of organismal processes that involve energy, is of critical importance in understanding the function and evolution ...

Ex Situ Culture of Pocillopora acuta and Effective Feeding Techniques

Effective Techniques for the Feeding and Ex Situ Culture of a Brooding Scleractinian Coral, Pocillopora acuta

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition expected in reef ecosystems over the next several decades. Recognition of this reef degradation has prompted a range of novel research- and restoration-based active interventions. Ex situ aquaculture can play a supporting role through the establishment of robust coral culture protocols (e.g., to improve health and reproduction in long-term experiments) and through the provision of a consistent broodstock supply (e.g., for use in restoration projects). Here, simple techniques for the feeding and ex situ culture of brooding scleractinian corals are outlined using the common and well-studied coral, Pocillopora acuta, as an example. To demonstrate this approach, coral colonies were exposed to different temperatures (24 &#176;C vs. 28 &#176;C) and feeding treatments (fed vs. unfed) and the reproductive output and timing, as well as the feasibility of feeding Artemia nauplii to corals at both temperatures, was compared. Reproductive output showed high variation across colonies, with differing trends observed between the temperature treatments; at 24 &#176;C, fed colonies produced more larvae than unfed colonies, but the opposite was found in colonies cultured at 28 &#176;C. All colonies reproduced before the full moon, and differences in reproductive timing were only found between unfed colonies in the 28 &#176;C treatment and fed colonies in the 24 &#176;C treatment (mean lunar day of reproduction &#177; standard deviation: 6.5 &#177; 2.5 and 11.1 &#177; 2.6, respectively). The coral colonies fed efficiently on Artemia nauplii at both treatment temperatures. These proposed feeding and culture techniques focus on the reduction of coral stress and the promotion of reproductive longevity in a cost-effective and customizable manner, with versatile applicability in both flow-through and recirculating aquaculture systems.

Author Spotlight: Advancing Coral Research by Exploring Climate Change Resistance, Ex Situ Aquaculture, and Reproduction Strategies 

Climate change is impacting coral reef ecosystems globally. Corals sourced from ex situ aquaculture systems can help support restoration and research efforts. Herein, feeding and coral culture techniques that can be used to promote the long-term maintenance of brooding scleractinian corals ex situ are outlined.

Climate change is affecting the survival, growth, and recruitment of corals globally, with large-scale shifts in abundance and community composition ...

Metabolite Profiling of Hard Coral Samples Using GC-MS

Gas Chromatography-Mass Spectrometry-Based Targeted Metabolomics of Hard Coral Samples

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the cnidarian-dinoflagellate symbiosis and how coral responds to stress (i.e., during temperature-induced bleaching). Steady-state metabolite profiling of the coral holobiont, which comprises the cnidarian host and its associated microbes (Symbiodiniaceae and other protists, bacteria, archaea, fungi, and viruses), has been successfully applied under ambient and stress conditions to characterize the holistic metabolic status of the coral.
However, to answer questions surrounding the symbiotic interactions, it is necessary to analyze the metabolite profiles of the coral host and its algal symbionts independently, which can only be achieved by physical separation and isolation of the tissues, followed by independent extraction and analysis. While the application of metabolomics is relatively new to the coral field, the sustained efforts of research groups have resulted in the development of robust methods for analyzing metabolites in corals, including the separation of the coral host tissue and algal symbionts.
This paper presents a step-by-step guide for holobiont separation and the extraction of metabolites for GC-MS analysis, including key optimization steps for consideration. We demonstrate how, once analyzed independently, the combined metabolite profile of the two fractions (coral and Symbiodiniaceae) is similar to the profile of the whole (holobiont), but by separating the tissues, we can also obtain key information about the metabolism of and interactions between the two partners that cannot be obtained from the whole alone.

Author Spotlight: Separation of Coral Host Tissues and Algal Symbionts and Analyzing Their Metabolites 

Here, we present the extraction and preparation of polar and semi-polar metabolites from a coral holobiont, as well as separated coral host tissue and Symbiodiniaceae cell fractions, for gas chromatography-mass spectrometry analysis.

Gas chromatography-mass spectrometry (GC-MS)-based approaches have proven to be powerful for elucidating the metabolic basis of the ...

Optimizing Coral Sperm Cryopreservation from Threatened Coral Species

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

Author Spotlight: Developing Efficient Cryopreservation and Biobanking Technologies for Global Reef Restoration

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around ...

Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video